Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Interactions between cyclic adenosine monophosphate (cAMP) and Ca2+ are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP3R) to IP3. We show that PTH communicates with IP3R via “cAMP junctions” that allow local delivery of a supramaximal concentration of cAMP to IP3R, directly increasing their sensitivity to IP3. These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP3R, but IP3R2 and AC6 are specifically associated, and inhibition of AC6 or IP3R2 expression by small interfering RNA selectively attenuates potentiation of Ca2+ signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP3R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

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In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC 50 of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr 494 and Ser 498 . Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr 494 and Ser 498 to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.The NADPH oxidases or Noxs define a unique family of enzymes that exist to synthesize reactive oxygen species (ROS) 2 (1). Currently, five distinct Noxs have been identified and are numbered Nox1 to -5. Of these, Nox2 is the best characterized and is expressed primarily by cells of the immune system, including neutrophils and macrophages. Mutation of Nox2 or targeted disruption in mice results in impaired host defense (2). The biological functions of Noxs that are expressed in cells outside of the immune system are emerging but remain somewhat enigmatic. However, the considerable diversity that exists in the location, the capacity, and the mechanisms by which these enzymes are activated suggests that they have indeed evolved distinct functional roles.The mechanisms that control the synthesis of ROS by the various Nox isoforms are remarkable in their complexity. Nox2 is a transmembrane glycoprotein that is activated by the coordinated assembly of at least five distinct subunits. In unstimulated cells, Nox2 is bound to the protein p22 phox , and upon activation, the coalescence of p67 phox , p47 phox , p40 phox , and Rac yields a functional, superoxide-generating oxidase (1, 3). Nox1 is also bound to p22 phox and is activated by the subunits NOXA1 and NOXO1, which are functionally related to p67 phox and p47 phox , respectively (4, 5). The activity of Nox3 can be regulated by p2...

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.The endothelial nitric-oxide synthase (eNOS), 4 which catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), is posttranslationally regulated by diverse protein-protein interactions and by covalent modification with fatty acylation and phosphorylation. eNOS, like the other two NOS isoforms termed neuronal NOS and inducible NOS, is a homodimer, with each of the two subunits having a bidomain structure consisting of an N-terminal oxygenase domain containing a heme moiety and binding sites for zinc, arginine, and the cofactor, tetrahydrobiopterin, and a C-terminal reductase domain that contains binding sites for FAD, FMN, and NADPH (1). Located between the oxygenase and reductase domains is a Ca 2ϩ calmodulin (CaM) binding sequence (2). Five serine/threonine sites of phosphorylation of bovine eNOS have been identified at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (3-10). eNOS phosphorylation at Ser-1179 increases eNOS activity by increasing the rate of electron flux through the reductase domain (11). Based on mutagenesis experiments, phosphorylation at Ser-635 increases the maximal activity of eNOS by an as yet unknown mechanism, whereas phosphorylation at Ser-617 does not alter maximal activity but significantly increases the Ca 2ϩ -CaM sensitivity of the enzyme (8). In contrast, eNOS phosphorylation at Thr-497 within the CaM binding sequence reduces eNOS catalytic activity by decreasing the binding affinity for Ca 2ϩ -CaM (7). The function of eNOS phosphorylation at Ser-116 is currently not well understood (12).Evidence exists that eNOS may also be regulated by tyrosine phosphorylation. have reported that treatment of endothelial cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, or the oxidant, hydrogen peroxide (H 2 O 2 )...